Bottom Line:
Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion.Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites.By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.

ABSTRACTEukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion. In the budding yeast Saccharomyces cerevisiae, the activation times of individual origins are not intrinsic to those origins but are instead governed by surrounding sequences. Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites. By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.

Mentions:
Determination of the initiation bias in the two-ARS plasmid provides a relatively simple assay for a mutational analysis of this element. Previous observations showed that deletion of the NcoI-NruI fragment, which included the 5′ half of URA3 (plasmid pΔ34), does not eliminate the preferential use of ARS1S, but that deletion of the NsiI-NcoI fragment from the 3′ end of URA3 (plasmid pΔ43) results in equivalent use of both copies of ARS1 (Figure 2; Brewer and Fangman 1994). To further localize the early determinant, four deletions (plasmids pΔ5, pΔ6, pΔ7, and pΔ8) that remove sections of the NsiI-NcoI sequence were constructed using convenient restriction sites, and tested for origin bias. Deletion of the 169-bp ApaI-NcoI fragment (pΔ5), the 302-bp AlwNI-ApaI fragment (pΔ6), or the 113-bp HaeIII-AlwNI fragment (pΔ7) had no significant reduction of the bias. However, the 34-bp NsiI-HaeIII fragment contributes substantially to the bias because its deletion (pΔ8) significantly reduced the bias (Figure 2).

Mentions:
Determination of the initiation bias in the two-ARS plasmid provides a relatively simple assay for a mutational analysis of this element. Previous observations showed that deletion of the NcoI-NruI fragment, which included the 5′ half of URA3 (plasmid pΔ34), does not eliminate the preferential use of ARS1S, but that deletion of the NsiI-NcoI fragment from the 3′ end of URA3 (plasmid pΔ43) results in equivalent use of both copies of ARS1 (Figure 2; Brewer and Fangman 1994). To further localize the early determinant, four deletions (plasmids pΔ5, pΔ6, pΔ7, and pΔ8) that remove sections of the NsiI-NcoI sequence were constructed using convenient restriction sites, and tested for origin bias. Deletion of the 169-bp ApaI-NcoI fragment (pΔ5), the 302-bp AlwNI-ApaI fragment (pΔ6), or the 113-bp HaeIII-AlwNI fragment (pΔ7) had no significant reduction of the bias. However, the 34-bp NsiI-HaeIII fragment contributes substantially to the bias because its deletion (pΔ8) significantly reduced the bias (Figure 2).

Bottom Line:
Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion.Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites.By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.

ABSTRACTEukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion. In the budding yeast Saccharomyces cerevisiae, the activation times of individual origins are not intrinsic to those origins but are instead governed by surrounding sequences. Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites. By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.